1. Field of the Invention
The present invention relates generally to analysis such as cytopathology and more particularly to methods and apparatus for utilizing dielectrophoresis (DEP) to achieve improved analysis techniques such as cytopathology techniques. Even more particularly, the present invention provides for the generation of segregated analysis smears that can be used in cytopathology and other applications (which the inventors have coined “electrosmears” or “electrosmear cytopathology”).
2. Background
Cytology slides are prepared to screen and diagnose cellular samples taken from, for example, tissue samples, samples from the uterine cervix, urine, sputum, blood, fine needle aspiration biopsy, urethral, bronchial brushings and washings, cerebral spinal fluid, and other body fluids. The reliability and efficacy of the screening methods of these slides are measured by their ability to diagnose infections, precancerous lesions or cancerous lesions while at the same time avoiding false positive or negative diagnosis. The reliability of these slides is a primary issue. Often, the results are not accurate or are unreadable. Thus, there is a constant effort to improve the reliability and efficacy in the preparation of cytology samples.
One of the most common uses of cytology slides is for screening and diagnosis of a cervical sample. Carcinoma of the cervix is one of the most common malignancies in women, causing thousands of deaths per year in the United States. A large proportion of these cases are associated with absent or deficient screening, and many screening failures are the result of errors in cervical sampling or smear interpretation.
Screening for precancerous or cancerous changes of the uterine cervix traditionally involves microscopic assessment of cervical Papanicolaou smears, called Pap smears. This traditional method for screening requires scraping a woman's cervix with a sampling device, such as a cotton applicator stick, spatula or brush, and smearing this sample onto a slide for review by a medical lab professional. The specimen is gently spread across a slide to evenly distribute the cell sample. On the slide itself, cells of interest do not necessarily follow any recognizable geometrical arrangement; rather, they are arranged randomly. In other words, (a) it just as likely that a precancerous cell may be found near the center of the slide versus near the left end of the slide versus near the right end of the slide and (b) cells of interest are not necessarily grouped together or separated from other groups of cells for easy identification. Following the formation of the smear, the slide is fixed, stained, and examined under a light microscope for cellular abnormalities.
In carrying out this operation, the portion of the sample that is smeared onto the slide may contain blood, mucus, inflammatory cells, and clumps of cells. Accurate interpretation of up to 40% of conventional Pap smears are compromised by the presence of blood, mucous, obscuring inflammatory cells, scant cellular material, and air-drying artifacts. The presence of these contaminants can obscure many of the cells, causing important precancerous lesions to be missed when the slide is reviewed at the lab or, alternatively, making the entire slide unreadable. Techniques that attempt to more effectively distribute matter within the sample onto a slide typically utilize spinning, which, although it improves screening somewhat, still yields a randomized, non-segregated distribution of cellular components.
Accordingly, one of the problems with conventional cytopathology techniques is the inability to create adequately segregated smears where cells of interest may be grouped apart from other cells. Because conventional smears are effectively random (i.e., the cells of interest do not necessarily follow any recognizable grouping or segregation pattern), important features of the sample may be obscured and/or completely overlooked. This overlooking of features may, in turn, lead to deficient screening. When a clinician is presented with a conventional, randomly-distributed smear, it may be difficult to effectively analyze that sample. In particular, analyzing a sample having a randomized distribution would be more difficult and time-consuming than analyzing a sample whose cells of interest were grouped together, apart from other less important cells.
Another problem with the conventional Pap smear is the frequent inaccuracy of the test result. Common inaccuracies include both false positive and false negative Pap test results. A false positive Pap test occurs when a patient is told she has abnormal cells when the cells are actually normal. A false positive result may require a woman to undergo unnecessary and costly medical procedures. A false negative Pap test result occurs when a specimen is called normal, but the woman has a lesion. A false negative Pap test may delay the diagnosis and treatment of a precancerous or even a cancerous condition.
The conventional Pap smear has false negative rates ranging from 10–50%, with up to 90% of those false negatives due to limitations of sampling or slide preparation. To decrease false negative rates associated with interpretation error, re-screening a portion of the negative smear or recalling the patient for another sample is often required.
Concern over the frequency of false-negative results of the traditional Pap smear has led to the development of a variety of other technologies or clinical strategies, such as liquid-based cytology systems, to improve Pap testing. For example, the Cytyc, Inc. (Marlborough, Mass.), ThinPrep® and the TriPath, Inc. (Burlington, N.C.), CytoRich® Pap test systems are two commercially available, FDA approved fluid-based methods used for the collection and preparation of cervicovaginal samples.
With the ThinPrep® system, a gynecologic sample is collected in the same manner as the conventional Pap test using a broom-type device or plastic spatula and endocervical brush combination, but rather than smearing the cytological sample directly onto a microscope slide, this method suspends the sample cells in a fixative solution (i.e. PreservCyt®). The ThinPrep® slide preparation system uses an automated apparatus called a Cytyc 2000® that involves filtration using vacuum pressure and positive pressure-transfer steps to prepare cytology slides.
With the CytoRich® slide preparation system, the gynecologic sample is also collected in the same manner as the conventional Pap test. Like the ThinPrep® system, the CytoRich® system also places the sample in a liquid medium for further purification prior to analysis. CytoRich® specimens are processed using two centrifugation steps through a gradient solution to separate the diagnostic cells from the interfering material. The cells are ultimately re-suspended in a final preparation that is applied to the slide using a special pipetting apparatus (Autocyte Prep System®) provided by the manufacturers (Tripath, Inc.). This transfer step can also be performed manually. Thereafter, a sample is placed on a slide and analyzed by cytology.
These new methods have demonstrated increased quality in the preparation of the sample, improved detection rates, and a reduced need for patients who must return for repeat smears. However, in both the ThinPrep® and the CytoRich® slide preparation systems, a time consuming and expensive procedure is followed to prepare a mono-dispersed layer of cells on a cytology slide. Additionally, despite their improvements, these systems are still not able to provide segregated smear samples, the presence of which would lead to more effective screening techniques.
In other cytological analyses, it is important to identify small numbers of diagnostically indicative cells within an overwhelmingly large concentration of background cells. For example, tumor cells may occur as a highly rarified subpopulation dispersed amongst normal cells in peripheral blood at concentrations below 1 tumor cell per 106 nucleated blood cells. Similarly, rarified tumor cells may occur amongst lymph and blood cells in biopsies taken from lymph nodes proximal to a tumor. Such cells are of importance to the detection, prognosis and treatment of cancers. Also, the peripheral blood of a pregnant woman contains a very small concentration of fetal cells. Isolation and analysis of these can facilitate the identification of fetal status without the need for potentially risky in utero biopsy procedures. In other cases, disease states may be associated with a very small concentration of yeast, viral or bacterial cells mixed with blood, sputum, urine, or other suspensions of cells and particulate debris. Banding and identification of such pathogens, which is not offered by conventional cytopathology techniques, is of profound importance to disease diagnosis.
As an additional example, biowarfare agents may be present against a background of other cells types such as blood, yeast, harmless bacteria or viruses as well as of debris and particulates including smoke, dust, pollen and other matter. The isolation and identification of such biowarfare agents is of importance to detecting acts of biological warfare and terrorism. The concentration, isolation, and analysis of rare subpopulations of such exemplary cell types and of others are of fundamental importance to both research, clinical practice, agriculture, and defense. However methods to capture rare cells in well-defined locations of a slide where they may be stained, readily identified, and analyzed by a pathologist or through scanning cytometry using, for example, staining, histochemical, and molecular methods, are lacking.
In some cases, the total number of cells in a sample may be very small and the use of conventional methods to prepare slides may result in significant sample loss as well as slides having such a widely dispersed distribution of indicative cells that the slides may be of poor diagnostic value. Methods that can capture very small numbers of cells from small samples within concentrated, well-defined, and precisely located bands are therefore desirable but lacking in conventional cytopathology techniques. Such methods would also be of important use as adjuncts to other cell sorting or fractionation methods in which defined cell subpopulations need to be captured and analyzed with minimal sample loss.
In sum, conventional cytopathology systems suffer from several shortcomings, one of the most prevalent being the inability to generate a segregated smear having distinct groupings of cells so that a clinician may better analyze the sample and provide quick, accurate, reliable screening and/or diagnosis.
The referenced shortcomings are not intended to be exhaustive, but rather are among many that tend to impair the effectiveness of previously known techniques concerning cytopathology; however, those mentioned here are sufficient to demonstrate that methodology appearing in the art have not been altogether satisfactory and that a significant need exists for the techniques described and claimed in this disclosure.